|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1993 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1992 : ¥700,000 (Direct Cost : ¥700,000)
The relation of oncogenes and suppressor genes to cell culture was described.
First, gene amplification was analyzed in 23 cell lines derived from human esophageal squamous cell carcinoma (ESC) tissues by Southern and Northern hybridizations to c-myc, c-erbB,hst-1, k-ras, b-FGF,CyclinD1. These genes showed no potential for establishing cell lines, with the exception of CyclinD1 expression which demonstated a slight potential to establish cell lines in a protein free environment.
Secondly, we performed allelotype analysis to investigate whether tumor suppressor genes are involved in esophageal cancer. Frequent loss of heterozygosity (LOH) of 30%>of the informative cases was observed on chromosomes 3p, 5q, 6p, 8p, 9p, 9q, 11p, 13q, 17p, 17q, 18q, and 19q. Among these, LOH on 5q, 13q, 17p, and 18q was considered to involve the APC,RB,p53, and DCC.However, deletion analysis of chromosome 18q revealed that the region commonly lost did not include the DCC locus. Screening of 60 primary ESC tum
ors and 20 cultured ESC cell lines for the mutation of the APC gene revealed that there was not any mutation despite the high frequency of LOH on chromosome 5q.
Thirdly, we screened 29 human ESC cell lines for mutations of the p53 gene through all coding exons and non-intron junctions. Mutations were found in 22 cell lines (76%) . We also examined 65 fresh tumor samples, from all of which we tried to establish cell lines, and detected mutations in 26 samples (40%) . There was no significant correlation between the status of the p53 gene in fresh tumors and the establishment of cell lines. We conclude that p53 mutation is not important in establishing cultured human ESC cell lines but provides a growth advantage in vitro. Neither was a relationship observed between p53 mutation and cell growth in protein free environment.
We also examined relationships between p53 mutation, murine double minute 2 (MDM2) amplification, and human papillomavirus (HPV) infection in ESC.In most tumors, amplification of the MDM2 gene or infection of HPV was not associated with p53 mutations. In cell culture, MDM2 gene amplification only had a significant relation to cell culture.
With regard to patients prognosis, CyclinD1 gene amplification was significantly correlated to poor prognosis in patients and we also found that a group of patients with p53 mutation and/or MDM2 amplification exhibited a significantly shorter survival period.
We are now studying the relationship of invasive character and cell membrane fluidity to protein free culture. Less